穿心莲内酯诱导胃癌细胞周期抑制和内源性凋亡

Andrographolide induces cycle arrest at G2/M phase and intrinsic apoptosis in gastric carcinoma cells

研究了穿心莲内酯诱导胃癌细胞SGC-7901生长抑制、周期阻滞与细胞凋亡的作用机制.用梯度浓度的穿心莲内酯作用于SGC-7901细胞,以MTT法检测药物对细胞的生长抑制情况;以流式细胞技术检测SGC-7901的周期分布状况、胞内的ROS水平以及线粒体膜电位的变化;以Annexin V-FITC/PI双染法检测细胞凋亡的水平;以Western blot检测不同加药浓度处理后的细胞内相关蛋白水平的变化.可知穿心莲内酯对SGC-7901细胞具有时间依赖和浓度依赖的抑制效果,48h的IC50约为24μg·mL-1;并且能够有效抑制G2/M期的周期进展,随后诱发内源性凋亡途径;与此同时,细胞内的ROS水平上升,线粒体膜电位下降;再加入NADPH之后,细胞内的GSH水平有所回升,周期阻滞得到缓解.细胞内的p53、p-CDC2、cleaved caspase-3/9、Bax等上调;bcl-2等水平下调.穿心莲内酯是通过上调胞内ROS水平并使线粒体膜电位崩溃而诱发G2/M期的周期阻滞与内源性凋亡,最终抑制SGC-7901细胞的生长.

Andrographolide is one of the extractions fromAndrographis paniculata,which is a traditional herb early reported its anti-tumor efficacy,and is proved it's the active ingredient which could suppress multiple carcinoma.It is proclaimed that many kinds of mechanisms concentrated on autophagy or apoptosis,and correlative signal pathway.To verify whether andrographolide would prevent amplification of gastric cancer cells,and to investigate the mechanism of andrographolide's effect on cell growth,cycle arrest and induction of apoptosis in vitro,we designed the experiment:SGC7901 was treated with different doses(0,16,24 and 32μg· mL^(-1))of andrographolide for 24,48 and 72h;Cell viability was measured by MTT assays to determine the efficacious time of performance.Then,investigations of cell cycle,ROS level and mitochondria membrane potential were proceeded and detected by flow cytometry.Cell apoptosis was monitored by flow cytometry using Annexin V-FITC/PI staining.Expression level of p53,p-CDC2,bcl-2,Bax were tested by western blot.Finally,we found that andrographolide effectively inhibited SGC-7901 cell growth and the IC50 was 24μg· mL^(-1) in 48 h.Meanwhile,the medicine showed the ability of cell cycle arrest at G2/M phase and apoptosis above 60% when treated with 24μg· mL^(-1)andrographolide for 48 h.Western blot indicated that cell cycle and apoptosis relevant proteins,such as p53,p-CDC2 and Bax were up-regulated and bcl-2 was down-regulated.In the end,we conclude that andrographolide inhibits SGC-7901 cell proliferation through G2/M phase arrest and inducing intrinsic apoptosis pathway.